The most general techniques for imaging specific proteins within cells and organisms rely either on antibodies or genetic tags. Electron microscopy (EM) is the standard technique for ultrastructural localization, but conventional EM immunolabeling remains challenging because of the need to develop high-affinity, high-selectivity antibodies that recognize cross-linked antigens. Furthermore, the optimal preservation of ultrastructure and visibility of cellular landmarks in EM requires strong fixation that hinders diffusibility of antibodies and gold particles. Thus the target proteins most easily labeled are those exposed at cut tissue surfaces. Replacement of bulky gold particles by eosin enables catalytic amplification via photooxidation of diaminobenzidine (DAB), but eosin-conjugated macromolecules still have limited diffusibility and need detergent permeabilization to enter cells [1]. Genetic labeling methods should overcome many of these shortcomings, just as fluorescent proteins have revolutionized light microscopic imaging in molecular and cell biology [2]. However, no analogous genetically encoded tag for EM contrast has yet proven widely applicable.
Metallothionein has been proposed as a genetic tag that can noncatalytically incorporate cadmium or gold [3], but its main applications to intact cells have been to Escherichia coli conditioned to tolerate 0.2 mM CdCl2 for 18 h [4] or 10 mM AuCl for 3 h [4], [5]. Such high concentrations of heavy metal salts would not seem readily transferable to most multicellular organisms or their cells. Also many higher organisms express endogenous metallothionein, which would contribute background signals unless genetically deleted or knocked down [5].
Horseradish peroxidase can be a genetic label in the secretory pathway but is greatly limited by its requirements for tetramerization, glycosylation, and high Ca2+, so that it is not functional when expressed in the cytosol [6]. Furthermore, its DAB reaction product tends to diffuse from sites of enzymatic generation, resulting in poorer resolution than immunogold or the reaction product of photogenerated singlet oxygen (1O2, the metastable excited state of O2) with DAB [1], [7], [8].
Controlled local photogeneration of singlet oxygen (1O2, the metastable excited state of O2) is useful for generating electron-microscopic contrast, rapidly inactivating proteins of interest, reporting protein proximities over tens of nanometers, and ablating cells by photodynamic damage. The best previous genetically targetable generator of 1O2 is the biarsenical dye ReAsH, which binds to genetically appended or inserted tetracysteine motifs [9]. However, ReAsH has modest 1O2 quantum yield (0.024), requires antidotes to prevent cell toxicity, needs careful precautions to reduce nonspecific background signal, and has been difficult to apply to multicellular tissues and organisms [10].
Although fluorescence photooxidation using GFP has been reported [11], [12], the 1O2 quantum yield of the naked GFP chromophore is extremely low (0.004), and the 1O2 quantum yield of the intact protein was yet lower and unquantifiable [13], presumably because the beta-barrel of the protein shields the chromophore from oxygen. The phototoxic fluorescent protein “Killer Red” [14] is now acknowledged not to work through 1O2 [15], and we have confirmed that its 1O2 quantum yield is negligible.
As such, a need exists for reagents and methods that can be used to image the ultrastructural localization of proteins in cells and tissues. Specifically, there exists a need for a genetically encoded tag that can be used to enhance the EM contrast of a specific protein in a fixed tissue sample without requiring the diffusion of a large molecule, such as an antibody or gold particle, into the tissue.